Citations

Abstract Introduction It is critical for the immune system to destroy cells harboring intracellular infection to remove pathogen burden. This function is widely attributed to cytotoxic T cells, which can physically engage with their targets through the T cell receptor complex, releasing cytotoxic granules, resulting in a specific death of infected targets. However, effector cytokines tumor necrosis factor-α (TNF) and interferon-γ (IFNγ), secreted by T cells, are themselves cytotoxic in combination to a degree. While TNF has appreciated cell death function through the TNF receptor 1 (TNFR1) signaling pathway, no clear cytotoxic function is attributed to IFNγ. Because of the diffusible nature of these cytokines, it is possible for T cells to elicit cytotoxicity without requiring direct contact with their targets, termed paracrine killing. Whether paracrine killing occurs during viral infection and how it is regulated is unclear. Paracrine killing poses issues of both sensitivity to these cytokines in that high concentrations of these cytokines are needed to affect target cells, as well as specificity in discriminating infected from uninfected host cells. Accounting for both issues, we hypothesize that paracrine killing is elicited through pathogen restriction whereby only infected cells are susceptible to death. Methods Analysis was performed in vitro using the Incucyte live cell imaging system and Western blots with fibroblast targets and a VSV infection model and OT1 T cells were leveraged ex vivo. Results TNF and IFNγ protect cells against the cytopathic effect of the virus. TNF specifically synergizes with VSV resulting in death of the infected cells and the two cytokines have distinct roles that work in concert. We demonstrate that TNF and IFNγ released by activated T cells suppress viral infection of nearby cells in a paracrine manner. Conclusion Here we provide insight and context into how distinct cytokines function in a cooperative manner for immune protection during an infection. Funding Source Mayo Clinic Graduate School of Biomedical Sciences Topic Categories Viral Immunology (VIR)

Cytotoxic T cells produce the cytokine interferon-γ (IFN-γ). We sought to determine whether IFN-γ directly kills target cells and found that T cells used IFN-γ to kill murine melanoma cells lacking the kinases TBK1 and IKKε. In the absence of both kinases, IFN-γ induced the production of TNF receptor 1 (TNFR1) and the sensor Z-DNA binding protein 1 (ZBP1) in the tumor cells to stimulate receptor-interacting protein kinase 1 (RIPK1)–dependent apoptosis in a cell-autonomous manner. IFN-γ also enhanced the activation of nuclear factor κB (NF-κB) signaling in a TNFR1-dependent manner in cells deficient in both TBK1 and IKKε. Because IFN-γ–induced apoptosis occurred in a transcription-dependent manner with slow kinetics, STAT1 and NF-κB cooperated to activate the expression of inflammatory genes in the dying cells. Thus, IFN-γ–induced cell death was accompanied by an inflammatory signature in the absence of TBK1 and IKKε. TBK1 and IKKε not only mediated the induction of type I IFN but also inhibited RIPK1-dependent death and NF-κB–dependent inflammation. Therefore, we propose that these kinases may have gained these intertwined functions so that cells infected by pathogens that produce antagonists of TBK1 and IKKε are eliminated by IFN-γ–secreting T cells in an inflammatory manner to compensate for the inhibition of type I IFN production.

ABSTRACT Cytotoxic T lymphocytes (CTLs) are known to eliminate target cells through perforin-mediated, contact-dependent killing - a process limited by the number of effector T cells despite its serial nature. CTLs likely also possess a mass killing mechanism that can eliminate target cells more efficiently. Using CAR-T cells against B7H3 present on B16 tumor targets, we demonstrate that activated T cells can also kill targets in a perforin-independent manner by releasing diffusible TNF and IFNγ capable of killing nearby targets in a paracrine fashion even if they bear no antigen. Against an unperturbed target, paracrine killing is inefficient but is enhanced by the deletion of TNFR1 signaling molecules such as TAK1, HOIP, or TBK1/IKKε. Notably, these molecules are inhibited naturally by pathogen-encoded antagonists. Expression of a microbial-encoded antagonist, such as the Yersinia -encoded YopJ that antagonizes TAK1 or Ebola-encoded VP35 that antagonizes TBK1/IKKε, alters the target cell response to these cytokines from non-lethal to lethal. We propose that target cells are inherently resistant to killing by TNF and IFNγ, but the presence of a microbial factor alters this sensitivity, providing for the selective elimination of the infected cell while minimizing harm to uninfected bystander cells. We propose the term ‘pathogen-restriction’ to describe this discriminatory mechanism. Potentially, any microbial-derived factor that crosstalks with the TNF and IFNγ signaling pathways can function as a pathogen-restriction element. GRAPHICAL ABSTRACT Short Summary T cells use TNF and IFNγ to kill target cells in a paracrine manner where target elimination is restricted by the presence of pathogen-derived factors.

Abstract Description Although contact-dependent, perforin-mediated cytotoxicity is crucial for T cell-mediated immunity, it is an inefficient method of eliminating unwanted cells. This is because the directional release of cytotoxic granules across the immunological synapse allows only one target cell to be killed at a time. This serial killing approach is likely insufficient at controlling rapid infections or an influx of malignant cells. One explanation for the poor effectiveness of CAR-T cells at treating solid tumors may be that the low efficiency contact-dependent killing of target cells require high effector:target (E:T) ratio may not be attainable clinically. Thus, developing strategies that enable T cells to kill tumor targets at a higher efficiency (i.e., lower E:T ratio) is warranted. Using a murine CAR-T system, we observed a direct role for T cell-derived cytokines in killing tumor target cells. As these effector molecules are diffusible, we found that this killing did not require effector-to-target contact but rather killed in a “remote” manner. Furthermore, we found that remote killing can be substantially improved if we inhibit TNF signaling molecules in the target cells that are often disabled by pathogens. Thus, tumor targets that are deficient in TAK1, HOIP or TBK1/IKKε can be killed by CAR-T cells at low E:T ratios. This work suggests that concurrently inhibiting certain TNF signaling molecules may be a strategy to augment CAR-T cell therapy for improved clinical outcome. Funding Sources T32A170478 Topic Categories Tumor Immunology: Checkpoints, Prevention, and Treatment (TIPT)

Abstract Description Antigen receptor (CAR) T cells can be generated in vivo within the natural lymph node (LN) environment as the immune response to a virus infection unfolds. This in situ approach entails priming LNs with intradermal inoculations of replication defective adenoviruses encoding MHC class I and class II allo/xeno-antigens. Universal transplant antigens were devised for LN priming by introducing tryptophan into amino acid position 162 of the MHC HLA-A2:01 class I heavy chain and by using the mouse MHC class II beta chain to form chimeric class II antigens with endogenous class II alleles. The virus encoded mismatched MHC antigen presenting molecules are expressed functionally in the LN following intradermal inoculation provoking polyclonal T cells responses. Three days later CAR T cells are generated by directly injecting retroviruses encoding CARs of interest into the responding LN. Mouse CD19-specifc CAR T cells created in this manner in B6-DQ8 and DQ8/A2 mice effectively target endogenous B cells and are therapeutic in male and female Emu-myc lymphomas demonstrating the potential for the generating functional CAR T cells directly in vivo. While in situ CAR T cells are readily produced in B6, B6-IA0/DQ8, B6-IA0/DQ8/A2, and B6-IA0/DQ6 LNs, functional differences among the strains emerged in the periphery suggesting that properties of the autoimmune-associated HLA DQ8 class II allele is particularly reactive during the production of CAR T cells in vivo. Funding Sources MustangBio, Inc Mayo Clinic Center of Biomedical Research Mayo Clinic Department of Immunology Mayo Clinic Graduate School of Biomedical Science Fraternal Order of Eagles Mayo Clinic Ventures NIH R21 AI 097337 NIH R01 CA 178804 NIH T32 AI 07425 Topic Categories Tumor Immunology: Checkpoints, Prevention, and Treatment (TIPT)

Abstract Chimeric antigen receptor (CAR) T-cells are T-cells engineered with synthetic receptors that directs their specificity and function toward an antigen of choice. This application has been highly successful in treating a variety of blood-borne cancers such as B-cell lymphomas. CAR T-cells against solid tumors have been less efficacious, potentially due to the resistance of tumor cells to T cell-mediated cytotoxicity. Current paradigm suggests that tumor cells are killed by perforin/granzyme-secreting CD8 cytotoxic T cells (CTLs). However, activated CD8 T cells also produce TNF and IFNg, two cytokines that have been reported to have anti-tumor function, but it is not clear whether these cytokines can directly kill tumor targets. It has been reported that defects in kinases in the TNFR1 signaling pathway can sensitize cells to TNF-induced death. Therefore, we hypothesize that by inactivating these kinases, we can sensitize solid tumors to killing by CD8 CAR T-cells producing TNF and IFNg. We intend to conduct proof-of-concept studies to show that sensitizing tumor cells to cytokine-mediated cytotoxicity will enhance the effectiveness of CAR T-cell therapy against solid tumors. Institutional Funding

Abstract T cells expressing chimeric antigen receptors (CAR T cells) are manufactured outside the body using non-physiological activation and in vitro culture protocols. The procedure takes 3 weeks and costs approximately $450,000 to administer. We have devised a scheme using mouse models for manufacturing CAR T cells in just 3 days employing a natural, polyclonal anti-viral immune response inside a lymph node (LN) using off the shelf reagents that can be applied widely in populations. CAR T cells emerge from the LN within a week, invade solid tumor, and eliminate the targeted antigen-positive tumor cells. Polyclonal activation of T cells is achieved by modifying the antigen presenting properties of lymph node resident professional antigen presenting cells (APC) through viral expression of allogeneic major histocompatibility antigens. The ensuing polyclonal activation occurs in the context of virus activated APC within the normal aarchitecture of the LN and confers robust T cell activation signals directing the development of cytolytic T cells. During the course of T cell activation, the T cells are transduced in situ with retroviruses encoding selected CARs. This approach could be applied to any number of different CARs to target a variety of tumors. T cells activated in the lymph node are functionally distinct from those activated using standard CAR T manufacturing protocols. We anticipate that CAR T cells generated in situ to natural stimuli will exhibit more normal immune regulatory properties that will impact their therapeutic utility. Because our approach uses off the shelf reagents and is administered using standard clinical procedures, cost to patients should be lowered and access to therapy increased.